Transplant patients sensitized to allo-Ag may undergo "accelerated" rejection (AccR), which combines cell and humoral immune mechanisms. We have developed a model of cardiac allograft rejection in skin sensitized mice (B/c- B6; rejection in <36h). We will study the role and mechanisms of CD154-CD4O T cell costimulation in host sensitization. These studies are important for: (i) further understanding the allograft rejection cascade in sensitized hosts; and (ii) defining novel mechanisms that may improve the long-term effects of CD154 blockade. We propose the following specific aims: (1). Analyze mechanisms by which CD154 blockade prevents AccR. Hypothesis: Inhibition of CD8+ T cell activation represents the principal mechanism of the CD154 blockade in sensitized hosts. First, we will use 2C TCR Tg system to dissect the mechanisms by which CD154 blockade prevents CD8+ T cell activation in sensitized hosts. Then, to test the functional significance of CD8+ T cell defect, we will compare the rejection process in sensitized WT hosts with or without CD4/CD8 effector branch depletion. Using T cell subsets or APCs alone or in combination from WT mice will test the restoration of graft rejection in CD154 KO recipients. We will determine whether activated CD8+ effectors (CD44high CD62Llow) alone can trigger accelerated rejection in naive CD154 KO hosts. We will delineate in vitro cross-talk between cell subsets after CD154 blockade that prevents CD8 T cell activation/differentiation. (2). Determine mechanisms by which CD1 54 blockade fails to prevent graft vasculopathy leading to chronic rejection. Hypothesis: Long-term allograft survival after CD154 blockade may be impaired by: (i) CD4-dependent afloreactive B cells, andior (ii) CD154-resistant or preexisting memory CD8 T cells. First, we will analyze serum alloreactive Ab levels and intragraft Ab deposition. The functional role of anti-donor Abs will be assessed in passive transfer studies (SCID). Then, we will determine the function of alloreactive B cells in vitro (ELISPOT) and in vivo (CD154 blockade in WT vs. B cell KO mice). The requirement of CD4 help for alloreactive B cells will be addressed in CD4 KO/CD4 mAbtreated WT mice. In parallel, we will study activation of resistant CD8+ and preexisting memory CD8+(CD44high CD62Llow T cells during the C0154 blockade by using GM1 marker and 2C ICR Tg system, respectively. (3). Investigate STAT4 vs STAT6 mediated mechanisms and the role of regulatory CD4+ T cells in CD154 blockade. Hypothesis: STAT6 signaling is important for the generation of regulatory CD4+ T cells to inhibit alloreactive residual CD8+ T and B cell responses after CD154 blockade. We will analyze the ability of CD154 blockade to affect early/ late allograft loss in WT, STAT4 vs. STAT6 KO mice. Proliferation, differentiation, and cytokine production by CD4+ and CD8+ I cells will be examined. We will establish the identity of regulatory CD4+ T cells, and determine their function in adoptive transfer experiments. STAT6-dependent mechanism will be further delineated after administration of regulatory CD4+ T cells into: (i) WT host + neutralizing lL-4/IL-10 mAb, and (ii) untreated STAT4 KO or STAT6 KO test mice.